tomato elongated petiole (tep-1): Shade Avoidance Mutant

Ellis Anderson
5/30/2016

Background slides courtesy of Leslie Herrera, May 2nd 2015

tep-1 Mutant

tep-1

  • Mutagenized seeds were screened for mutations
  • Found tep-1, a shade avoidance mutant
  • Characterized by constitutively elongated petioles

What is Shade Avoidance?

  • Low Red:Far-Red light is indicative of shade
  • Plants respond via
    • Internode and petiole elongation
    • Increased apical dominance
    • This is theorized to affect yield

Shade Avoidance

Research Question

What genes in domesticated tomato are involved in shade avoidance?

Elongated Mutant Screen

Wild-type: Standard for comparison

Wild Type Plants

Mutant of interest: Consitutive elongation

Mutant Plants

tomato elongated petiole (tep-1)

tep-1

Phenotype Characteristics

Extended Petioles

Mutant of interest (tep-1) has petioles that are longer than the control (M82) by the 5th week of development

Petiole

M82 vs tep-1 - Petiole

Finding the Gene: Bulk Segregant Analysis

A bioinformatics approach to find the mutation responsible for the tep-1 phenotype

Summary

BSA1

Summary

BSA2

Tissue Collection From Elongated F2 Plants

tep and heinz F2s

25% homozygous F2s

Sequencing and Computational Analysis

  • Filter low quality reads
  • Map reads to reference genome
  • Generate graphical output

illumina seq

Graphical Output to Locate Gene of Interest

  • Take advantage of different parental backgrounds to find region of interest
  • Should ideally produce one spot with 100% M82 background

Initial Results

Leslie's Results

My Work on tep-1

  • Check over Leslie's code and reproduce her results
  • Map Heinz genome to Heinz genome and add those results to the analysis
  • Look for deletions from fast neutron mutagenesis in region of interest

Recreating Leslie's Results

Run through Leslie's code step by step

Results: Results 1

Proofreading R Script

Filtering based on genotype

# Filter to keep rows where M82 = "1/1"
tep_snp_data <-subset(tep_data, tep.gt != "0/0")

tep_snp_data<-tep_snp_data[grep("TYPE=snp", tep_snp_data$INFO),]

tep_snp_data <- tep_snp_data[!is.na(tep_snp_data$tep.read.depth),]

Proofreading R Script

Filtering based on genotype

# Filter to keep rows where M82 = "1/1"
tep_snp_data <-subset(tep_data, tep.gt != "0/0")

tep_snp_data <- tep_snp_data[grepl("1/1",tep_snp_data$M82.gt),]

tep_snp_data<-tep_snp_data[grep("TYPE=snp", tep_snp_data$INFO),]

tep_snp_data <- tep_snp_data[!is.na(tep_snp_data$tep.read.depth),]

New Results

Results 2

Potential Causes For Multiple Peaks

  • Could be multiple genes causing tep-1 phenotype
    • That can be ruled out based on the 3:1 phenotype in F2s
  • Could be a bioinformatics issue

Reference Issues

Heinz-Heinz

Mapping Heinz to Heinz

  • Used heinz reads from Lavelle tie1 experiment: SRR404081.fastq.gz
  • Mapped these to S_lycopersicum_chromosomes.2.50.fa
  • Reads were mapped using bwa mem

Merging Heinz file to Leslie's

  • Used picard-tools functions to add read groups and combine all reads
  • Used freebayes to call SNPs for analysis in R

R analysis

Added additional filtering criteria to remove any Heinz-Heinz polymorphisms

tep_snp_data <- tep_snp_data[grepl("0/0",tep_snp_data$Heinz.gt),]

Final Results:

Chromosome 9: Ch 09 - Final

Final Results:

All Chromosomes: All Chromosomes - Final1

Final Results:

All Chromosomes: All Chromosomes - Final2

Next Steps

  • Area of about 300kb identified as 100% M82
    • 10 genes within this region
  • Fast neutron mutagenesis often leads to large deletions
  • Map reads again, but this time look for large deletions in the area of interest
  • Fast neutron mutagensis also induces SNPs
  • Look for SNPs causing missense or nonsense mutations in that interval

Genes in Region of Interest

Name Description
Solyc09g075980.2 Os01g0778500 protein
Solyc09g075990.2 IPR002828 Survival protein SurE-like phosphatase/nucleotidase
Solyc09g076000.2 Serine/threonine protein kinase
Solyc09g076010.2 IPR019787 Zinc finger, PHD-finger
Solyc09g076020.2 Imidazoleglycerol-phosphate dehydratase
Solyc09g076040.2 Protein SUPPRESSOR OF GENE SILENCING 3 homolog
Solyc09g076050.2 Frigida-like
Solyc09g082060.2 Cysteine synthase
Solyc09g082090.1 Unknown Protein
Solyc09g082080.1 Plant-specific domain TIGR01568 family protein

Likely Candidate Genes

Name Description
Solyc09g076000.2 Serine/threonine protein kinase
Solyc09g076040.2 Protein SUPPRESSOR OF GENE SILENCING 3 homolog
Solyc09g076010.2 IPR019787 Zinc finger, PHD-finger

MAPKKK71

  • Serine/threonine protein kinase was identified as MAPKKK71
  • MAPKKK71 is auxin inducible (Wu et al. 2014)

Wu et al. 2014

Suppressor of Gene Silencing 3

  • Arabadopsis homolog shown to interact with auxin pathway members
  • Affects leaf development (Adenot et al. 2006)

Further Work

  • Finish mapping for deletions
  • Search for nonsense or missense mutations
  • Use molecular biology techniques to ensure identified gene results in tep-1 phenotype

Special Thanks

  • Julin Maloof
  • Cody Markelz
  • Leslie Herrera & Amanda Schrager Lavelle
  • Zamir Lab
  • Maloof and Harmer Lab members

References

  1. Wu J, Wang J, Pan C, Guan X, Wang Y, Liu S, et al. (2014) Genome-Wide Identification of MAPKK and MAPKKK Gene Families in Tomato and Transcriptional Profiling Analysis during Development and Stress Response. PLoS ONE 9(7): e103032. doi:10.1371/journal.pone.0103032

  2. Xavier Adenot, Taline Elmayan, Dominique Lauressergues, Stéphanie Boutet, Nicolas Bouché, Virginie Gasciolli, Hervé Vaucheret, DRB4-Dependent TAS3 trans-Acting siRNAs Control Leaf Morphology through AGO7, Current Biology, Volume 16, Issue 9, 9 May 2006, Pages 927-932, ISSN 0960-9822, http://dx.doi.org/10.1016/j.cub.2006.03.035.